The introduction of foreign materials which is not membrane-permeable (for example nucleic acid molecules, chromosomes, organelles, nanoparticles, proteins, dyes or active pharmaceutical agents) into cells is a widespread cell biology problem. The targeted introduction of the substances into selected single cells within a cell population is particularly difficult.
Whereas easy-to-handle methods for permeating the cell membrane and, hence, methods widely used in laboratories, for example, electroporation (namely the transient permeabilization of the cell membrane by voltage pulses) or the use of liposomes (lipid vesicles containing the foreign material that is to be introduced and which coalesce with the cell membrane) as a rule act simultaneously and nonspecifically on a multitude of cells, until a short time ago virtually only the method of microinjection was available for the manipulation of single cells, a method that is costly in terms of the required equipment and highly demanding in terms of the handling. By this method, the foreign material is injected directly into the cell nucleus or the cytoplasm of the cell with the aid of a microcapillary. The method has an efficiency close to 100%. Only relatively few cells, however, can be handled within a practical length of time.
In past years, progress in laser nanosurgery has led to the development of laser-mediated permeabilization of the cell membrane (optoperforation, also referred to as laser perforation or photoperforation) to permit in this manner the introduction of foreign material into selected single cells. By means of an appropriate microscope arrangement, for example, as the one described by Stevenson et al. (2006, Optics Express, Vol. 14, No. 16, pp. 7125-33), the cell membrane of single cells can be irradiated by pulsed, laser radiation. At the site of the incidence of the laser radiation on the cell membrane, cavitation bubbles are formed provided the radiation intensity is sufficient. In such a case, the bubbles are generated in a nominally transparent medium by multiphoton absorption, the two-photon absorption playing an important role (see Stevenson et al.). Meanwhile, it is assumed that when single laser pulses or a series of pulses with a repetition rate of ≦1 MHz are used, the target cell can effectively take up the foreign material to be introduced only when these cavitation bubbles are formed during the irradiation (Vogel et al., 2005, Applied Physics B 81, pp. 1015-47). On the other hand, excessively high radiation doses and thus too large cavitation bubbles exert a negative effect on the viability of the target cell resulting subsequently in an increased mortality of the treated cells, which in turns has a deleterious effect on the efficacy of the method.
The application of the optimum radiation dose that ensures effective permeabilization of the cell membrane at the highest possible survival rate thus represents the crucial prerequisite for the success of the method. The optimum radiation dose thus depends to a high degree on the specimen to be irradiated. Depending on the type of cell or tissue, the physiological condition of the cells and the medium or environment surrounding the cells, the laser parameters must be adapted individually in each case, namely a calibration for the laser treatment is needed as is the monitoring of this treatment.
A possible indicator of the effects achieved by the laser application could be the size of the cavitation bubbles formed in which case, as proposed by Vogel et al. (2005, Applied Physics B, 81, pp. 1015-47), the bubble size can be determined by measuring the bubble oscillation time (namely the bubble lifespan). In that publication, the light scattering by the bubbles formed in the laser focus is mentioned as a possible approach to on-line monitoring of the bubble size or bubble lifespan, but it is not described, how this goal is to be achieved.
In DE 103 31 792 A1 is disclosed a laser with dosimetry control whereby the first appearance of bubbles within a tissue can be detected also interferometrically through the change in refractive index. This serves to modulate the laser performance so that the irradiation can be carried out mostly very closely above the bubble formation threshold. In this case, however, only the appearance of the bubbles is detected, and the determination of the lifespan of the bubbles and conclusions drawn therefrom concerning the bubble size are not described.
The dissertation of Jörg Neumann on “Microscopic Studies Concerning Laser-induced Bubble Formation and Bubble Dynamics on Absorbing Microparticles” (University of Lübeck, 2005) deals with linear absorption processes on microparticles (absorbers, for example pigments) such as those taking place in laser therapy of absorbing cell layers, particularly on the ocular fundus. An important objective of this study is to reveal the impact of these particles on the bubble dynamics at different energies of the bubble-inducing radiation. The study discloses the measurement of the lifespan of laser-induced microbubbles by means of the scattering of a test laser beam in the focus of the bubble-induced radiation. In this case, bubbles having a lifespan of about 100 ns are detected. The method, however, is not suitable for use in real time, and no conversion to bubble sizes and thus no complete evaluation of the damage inflicted on the cells by the bubbles is done. Although the dissertation also discusses on-line dosimetry control by the interferometric methods involving back-scattering, the applicability of this control is limited to bubbles with a size in the micrometer-range and is primarily intended for the detection of the appearance of such bubbles.
Whether quantitative measurement of the bubble lifespan by a transmitted radiation method (for example via scattering) in a nominally transparent medium without absorber particles, in which the bubbles are formed individually by muultiphoton absorption and can have a diameter clearly below 100 nm, is at all possible, particularly as a method for on-line monitoring, was hitherto not known.